Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute Muscle Injury.

Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.

Distinct muscle regenerative capacity of human induced pluripotent stem cell-derived mesenchymal stromal cells in Ullrich congenital muscular dystrophy model mice.

BACKGROUND: Ullrich congenital muscular dystrophy (UCMD) is caused by a deficiency in type 6 collagen (COL6) due to mutations in COL6A1, COL6A2, or COL6A3. COL6 deficiency alters the extracellular matrix structure and biomechanical properties, leading to mitochondrial defects and impaired muscle regeneration. Therefore, mesenchymal stromal cells (MSCs) that secrete COL6 have attracted attention as potential therapeutic targets. Various tissue-derived MSCs exert therapeutic effects in various diseases. However, no reports have compared the effects of MSCs of different origins on UCMD pathology. METHODS: To evaluate which MSC population has the highest therapeutic efficacy for UCMD, in vivo (transplantation of MSCs to Col6a1-KO/NSG mice) and in vitro experiments (muscle stem cell [MuSCs] co-culture with MSCs) were conducted using adipose tissue-derived MSCs, bone marrow-derived MSCs, and xeno-free-induced iPSC-derived MSCs (XF-iMSCs). RESULTS: In transplantation experiments on Col6a1-KO/NSG mice, the group transplanted with XF-iMSCs showed significantly enhanced muscle fiber regeneration compared to the other groups 1 week after transplantation. At 12 weeks after transplantation, only the XF-iMSCs transplantation group showed a significantly larger muscle fiber diameter than the other groups without inducing fibrosis, which was observed in the other transplantation groups. Similarly, in co-culture experiments, XF-iMSCs were found to more effectively promote the fusion and differentiation of MuSCs derived from Col6a1-KO/NSG mice than the other primary MSCs investigated in this study. Additionally, in vitro knockdown and supplementation experiments suggested that the IGF2 secreted by XF-iMSCs promoted MuSC differentiation. CONCLUSION: XF-iMSCs are promising candidates for promoting muscle regeneration while avoiding fibrosis, offering a safer and more effective therapeutic approach for UCMD than other potential therapies.

SRSF1 Is Crucial for Maintaining Satellite Cell Homeostasis During Skeletal Muscle Growth and Regeneration.

BACKGROUND: The splicing factor SRSF1 emerges as a mater regulator of cell proliferation, displaying high expression in actively proliferative satellite cells (SCs). In SRSF1 knockout mice (KO) generated via MyoD-Cre, early mortality and muscle atrophy are observed during postnatal muscle growth. Despite these findings, the precise mechanisms through which SRSF1 loss influences SCs' functions and its role in muscle regeneration remain to be elucidated. METHODS: To unravel the exact mechanisms underlying the impact of SRSF1 deficiency SC functions, we employed single-cell RNA sequencing (scRNA-seq) on a mononuclear cell suspension isolated from the newborn diaphragm of KO and control mice. Concurrently, we subjected diaphragm muscles to RNA-seq analysis to identify dysregulated splicing events associated with SRSF1 deletion. For the analysis of the effect of SRSF1 deletion on muscle regeneration, we generated mice with inducible SC-specific Srsf1 ablation through Pax7-CreER. SRSF1 ablation was induced by intraperitoneal injection of tamoxifen. Using cardiotoxin-induced muscle injury, we examined the consequences of SRSF1 depletion on SC function through HE staining, immunostaining and EdU incorporation assay. C2C12 myoblasts and isolated myoblasts were employed to assess stem cell function and senescence. RESULTS: Utilizing scRNA-seq analysis, we observed a noteworthy increase in activated and proliferating myoblasts when SRSF1 was absent. This increase was substantial, with the proportion rising from 28.68% in the control group to 77.06% in the knockout group. However, these myoblasts experienced mitotic abnormalities in the absence of SRSF1, resulting in cell cycle arrest and the onset of cellular senescence. In the knockout mice, the proportion of Pax7+ cells within improper niche positioning increased significantly to 25% compared to 12% in the control cells (n ≥ 10, p < 0.001). Furthermore, there was an observation of persistent cell cycle exit specifically in the Pax7+ cells deficient in SRSF1 (n = 6, p < 0.001). SRSF1 plays a pivotal role in regulating the splicing of Fgfr1op2, favouring the full-length isoform crucial for mitotic spindle organization. Disrupting SRSF1 in C2C12 and primary myoblasts results in multipolar spindle formation (p < 0.001) and dysregulated splicing of Fgfr1op2 and triggers cellular senescence. Consequently, adult SCs lacking SRSF1 initially activate upon injury but face substantial challenge in proliferation (n = 4, p < 0.001), leading to a failure in muscle regeneration. CONCLUSIONS: SRSF1 plays a critical role in SCs by ensuring proper splicing, maintaining mitotic progression and preventing premature senescence. These findings underscore the significant role of SRSF1 in controlling SC proliferation during skeletal muscle growth and regeneration.

Overexpression of CPT1A disrupts the maintenance and regenerative function of muscle stem cells.

The skeletal muscle satellite cells (SCs) mediate regeneration of myofibers upon injury. As they switch from maintenance (quiescence) to regeneration, their relative reliance on glucose and fatty acid metabolism alters. To explore the contribution of mitochondrial fatty acid oxidation (FAO) pathway to SCs and myogenesis, we examined the role of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme of FAO. CPT1A is highly expressed in quiescent SCs (QSCs) compared with activated and proliferating SCs, and its expression level decreases during myogenic differentiation. Myod1Cre-driven overexpression (OE) of Cpt1a in embryonic myoblasts (Cpt1aMTG) reduces muscle weight, grip strength, and contractile force without affecting treadmill endurance of adult mice. Adult Cpt1aMTG mice have reduced number of SC, impairing muscle regeneration and promoting lipid infiltration. Similarly, Pax7CreER-driven, tamoxifen-inducible Cpt1a-OE in QSCs of adult muscles (Cpt1aPTG) leads to depletion of SCs and compromises muscle regeneration. The reduced proliferation of Cpt1a-OE SCs is associated with elevated level of acyl-carnitine, and acyl-carnitine treatment impedes proliferation of wildtype SCs. These findings indicate that aberrant level of CPT1A elevates acyl-carnitine to impair the maintenance, proliferation and regenerative function of SCs.

Aerobic Exercise Protects against Cardiotoxin-Induced Skeletal Muscle Injury in a DDAH1-Dependent Manner.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a critical enzyme that regulates nitric oxide (NO) signaling through the degradation of asymmetric dimethylarginine (ADMA). Previous studies have revealed a link between the beneficial effects of aerobic exercise and the upregulation of DDAH1 in bones and hearts. We previously reported that skeletal muscle DDAH1 plays a protective role in cardiotoxin (CTX)-induced skeletal muscle injury and regeneration. To determine the effects of aerobic exercise on CTX-induced skeletal muscle injury and the role of DDAH1 in this process, wild-type (WT) mice and skeletal muscle-specific Ddah1-knockout (Ddah1MKO) mice were subjected to swimming training for 8 weeks and then injected with CTX. In WT mice, swimming training for 8 weeks significantly promoted skeletal muscle regeneration and attenuated inflammation, oxidative stress, and apoptosis in the gastrocnemius (GA) muscle after CTX injection. These phenomena were associated with increases in the protein expression of PAX7, myogenin, MEF2A, eNOS, SOD2, and peroxiredoxin 5 and decreases in iNOS expression in GA muscles. Swimming training also decreased serum ADMA levels and increased serum nitrate/nitrite (NOx) levels and skeletal muscle DDAH1 expression. Interestingly, swimming training in Ddah1MKO mice had no obvious effect on CTX-induced skeletal muscle injury or regeneration and did not repress the CTX-induced inflammatory response, superoxide generation, or apoptosis. In summary, our data suggest that DDAH1 is important for the protective effect of aerobic exercise on skeletal muscle injury and regeneration.

Impact of Extremely Low-Frequency Electromagnetic Fields on Skeletal Muscle of Sedentary Adult Mice: A Pilot Study.

Extremely low-frequency electromagnetic fields (ELF-EMFs) are ubiquitous in industrialized environments due to the continuous use of electrical devices. Our previous studies demonstrated that ELF-EMFs affect muscle cells by modulating oxidative stress and enhancing myogenesis. This pilot study investigated these effects on the skeletal muscles of sedentary adult mice, assessing physiological responses to ELF-EMF exposure and potential modulation by antioxidant supplementation. Male C57BL/6 mice were exposed to ELF-EMFs (0.1 or 1.0 mT) for 1 h/day for up to 5 weeks and fed a standard diet without or with N-acetyl-cysteine (NAC). The results showed transient increases in muscle strength (after 2 weeks of exposure at 1.0 mT), potentially linked to muscle fiber recruitment and activation, revealed by higher PAX7 and myosin heavy chain (MyH) expression levels. After ELF-EMF exposure, oxidative status assessment revealed transient increases in the expression levels of SOD1 and catalase enzymes, in total antioxidant capacity, and in protein carbonyl levels, markers of oxidative damage. These effects were partially reduced by NAC. In conclusion, ELF-EMF exposure affects skeletal muscle physiology and NAC supplementation partially mitigates these effects, highlighting the complex interactions between ELF-EMFs and antioxidant pathways in vivo. Further investigations on ELF-EMFs as a therapeutic modality for muscle health are necessary.

Hotspots and trends in satellite cell research in muscle regeneration: A bibliometric visualization and analysis from 2010 to 2023.

Background: The incidence of muscle atrophy or sports injuries is increasing with time and population aging, thereby attracting considerable attention to muscle generation research. Muscle satellite cells, which play an important role in this process, lack comprehensive literature regarding their use for muscle regeneration. Hence, this study aimed to analyze the hotspots and trends in satellite cell research from 2010 to 2023, providing a reference for muscle regeneration research. Methods: Studies on satellite cells' role in muscle regeneration from 2010 to 2023 were retrieved from the Web of Science Core Collection. Using CiteSpace and VOSviewer, we analyzed annual publications, authors and co-citing authors, countries and institutions, journals and co-citing journals, co-citing references, and keywords. Results: From 2010 to 2023, 1468 papers were retrieved, indicating an overall increasing trend in the number of annual publications related to satellite cells in muscle regeneration. The United States had the highest number of publications, while the Institut National de la Santé et de la Recherche Médicale was the institution with the most publications. Among journals, " PloS One" had the highest number of published papers, and "Cell" emerged as the most co-cited journal. A total of 7425 authors were involved, with Michael A. Rudnicki being the author with the highest number of publications and the most co-cited author. The most cited reference was "Satellite cells and the muscle stem cell niche." Among keywords, "satellite cells" was the most common, with "heterogeneity" having the highest centrality. Frontier themes included "Duchenne muscular dystrophy," "skeletal muscle," "in-vivo," "muscle regeneration," "mice," "muscle atrophy," "muscle fibers," "inflammation," " mesenchymal stem cells," and "satellite cell." Conclusion: This study presents the current status and trends in satellite cell research on muscle regeneration from 2010 to 2023 using bibliometric analyses, providing valuable insights into numerous future research directions.

Tubular Aggregates as a Marker of Aging in Skeletal Muscle.

Tubular aggregates (TA) are skeletal muscle structures that arise from the progressive accumulation of sarcoplasmic reticulum proteins, mainly with aging. Muscle regeneration plays a role in TA formation. TA quantification may aid in the evaluation of muscle aging and genetic muscle degeneration. TA form over time, appears in aging in normal murine muscles. TA reduction in injured conditions may be due to the degeneration-regeneration process in muscles, with loss of damaged muscle fibers and formation of new fibers that do not present protein aggregation. These new regenerated fibers do not improve the function capacity of the aged muscle. Here, we present a methodology for labeling and identifying tubular aggregates in muscle fibers and also the standardization of its quantification.

Profound cellular defects attribute to muscular pathogenesis in the rhesus monkey model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutations in the DMD gene. Muscle fibers rely on the coordination of multiple cell types for repair and regenerative capacity. To elucidate the cellular and molecular changes in these cell types under pathologic conditions, we generated a rhesus monkey model for DMD that displays progressive muscle deterioration and impaired motor function, mirroring human conditions. By leveraging these DMD monkeys, we analyzed freshly isolated muscle tissues using single-cell RNA sequencing (scRNA-seq). Our analysis revealed changes in immune cell landscape, a reversion of lineage progressing directions in fibrotic fibro-adipogenic progenitors (FAPs), and TGF-ß resistance in FAPs and muscle stem cells (MuSCs). Furthermore, MuSCs displayed cell-intrinsic defects, leading to differentiation deficiencies. Our study provides important insights into the pathogenesis of DMD, offering a valuable model and dataset for further exploration of the underlying mechanisms, and serves as a suitable platform for developing and evaluating therapeutic interventions.

Diarylpropionitrile-stimulated ERß nuclear accumulation promotes MyoD-induced muscle regeneration in mdx mice by interacting with FOXO3A.

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive degenerative disease of skeletal muscle, characterized by intramuscular inflammation, muscle regeneration disorder and replacement of muscle with fibroadipose tissue. DMD is caused by the absence of normal dystrophy. Impaired self-renew ability and limited differentiation capacity of satellite cells are proved as main reasons for muscle regeneration failure. The deficiency of estrogen impedes the process of muscle regeneration. However, the role of estrogen receptor ß (ERß) in muscle regeneration is still unclear. This study aims to investigate the role and the pharmacological effect of ERß activation on muscle regeneration in mdx mice. This study showed that mRNA levels of ERß and myogenic-related genes both witnessed increasing trends in dystrophic context. Our results revealed that treatment with selective ERß agonist (DPN, diarylpropionitrile) significantly increased myogenic differentiation 1 (MyoD-1) level and promoted muscle regeneration in mdx mice. Similarly, in mdx mice with muscle-specific estrogen receptor α (ERα) ablation, DPN treatment still promoted muscle regeneration. Moreover, we demonstrated that myoblasts differentiation was accompanied by raised nuclear accumulation of ERß. DPN treatment augmented the nuclear accumulation of ERß and, thus, contributed to myotubes formation. One important finding was that forkhead box O3A (FOXO3A), as a pivotal transcription factor in Myod-1 transcription, participated in the ERß-promoted muscle regeneration. Overall, we offered an interesting explanation about the crucial role of ERß during myogenesis.

TMEM16A regulates satellite cell-mediated skeletal muscle regeneration by ensuring a moderate level of caspase 3 activity.

It has been documented that caspase 3 activity is necessary for skeletal muscle regeneration, but how its activity is regulated is largely unknown. Our previous report shows that intracellular TMEM16A, a calcium activated chloride channel, significantly regulates caspase 3 activity in myoblasts during skeletal muscle development. By using a mouse line with satellite cell (SC)-specific deletion of TMEM16A, we examined the role of TMEM16A in regulating caspase 3 activity in SC (or SC-derived myoblast) as well as skeletal muscle regeneration. The mutant animals displayed apparently impaired regeneration capacity in adult muscle along with enhanced ER stress and elevated caspase 3 activity in Tmem16a-/- SC derived myoblasts. Blockade of either excessive ER stress or caspase 3 activity by small molecules significantly restored the inhibited myogenic differentiation of Tmem16a-/- SCs, indicating that excessive caspase 3 activity resulted from TMEM16A deletion contributes to the impaired muscle regeneration and the upstream regulator of caspase 3 was ER stress. Our results revealed an essential role of TMEM16A in satellite cell-mediated skeletal muscle regeneration by ensuring a moderate level of caspase 3 activity.

The Hox-based positional memory in muscle stem cells.

The skeletal muscle is a contractile tissue distributed throughout the body with various anatomical sizes, shapes and functions. In pathological conditions, such as muscular dystrophy, age-related sarcopenia and cancer cachexia, skeletal muscles are not uniformly affected throughout the body. This region-specific vulnerability cannot be fully explained by known physiological classifications, including muscle fiber types. Accumulating evidence indicates that the expression patterns of topographic homeobox (Hox) genes provide a molecular signature of positional memory, reflecting the anatomical locations and embryonic history of muscles and their associated muscle stem cells in adult mice and humans. Hox-based positional memory is not merely a remnant of embryonic development but is expected to be an intrinsic determinant controlling muscle function because recent studies have shown that aberrant Hox genes affect muscle stem cells. In this review, we discuss the concept of Hox-based positional memory, which may offer a new perspective on the region-specific pathophysiology of muscle disorders.

Integration of single-cell datasets depicts profiles of macrophages and fibro/adipogenic progenitors in dystrophic muscle.

Single-cell technologies have recently expanded the possibilities for researchers to gain, at an unprecedented resolution level, knowledge about tissue composition, cell complexity, and heterogeneity. Moreover, the integration of data coming from different technologies and sources also offers, for the first time, the possibility to draw a holistic portrait of how cells behave to sustain tissue physiology during the human lifespan and disease. Here, we interrogated and integrated publicly available single-cell RNAseq data to advance the understanding of how macrophages, fibro/adipogenic progenitors, and other cell types establish gene regulatory networks and communicate with each other in the muscle tissue. We identified altered gene signatures and signaling pathways associated with the dystrophic condition, including an enhanced Spp1-Cd44 signaling in dystrophic macrophages. We shed light on the differences among dystrophic muscle aging, considering wild type, mdx, and more severe conditions as in the case of the mdx-2d model. Contextually, we provided details on existing communication relations between muscle niche cell populations, highlighting increased interactions and distinct signaling events that these cells stablish in the dystrophic microenvironment. We believe our findings can help scientists to formulate and test new hypotheses by moving towards a more complete understanding of muscle regeneration and immune system biology.

Quantifying Muscle Regeneration: Activated Muscle Satellite Cells and New Regenerated Myofibers in Chronic and Acute Degeneration Models.

Regeneration is a remarkable characteristic of the skeletal muscle. Triggered by common lesions, regeneration is stimulated resulting in muscle fiber repair and restoration of muscle homeostasis in normal muscle. In genetic dystrophic muscle, the cycle of degeneration/regeneration is an endless loop that leads to impaired regeneration and substitution of muscle fibers by connective and adipose tissue, causing muscle weakness. Identification and characterization of muscle regeneration steps can help discover potential therapy targets for muscle diseases and aging. Muscle regeneration markers such as the number of satellite cells in the muscle, the proportion of activated satellite cells, and the quantity of regenerating muscle fiber can be quantified using immunolabeling.Here we are presenting a quantitative method to measure muscle regeneration that can be applied to different proposals. To demonstrate the protocol applicability, we used models for acute and chronic muscle injuries. As model of acute degeneration, a wild-type C57BL6 mice with muscle injury induced by electroporation was used, and the muscle was analyzed after 5 and 10 days post-injury. DMDmdx mouse muscle was used as a model of chronic degeneration. The methodologies presented here are among the gold standard methodologies for muscle regeneration analysis and can be easily applied to any type of muscle regeneration study.

IL-33 Facilitates Fibro-Adipogenic Progenitors to Establish the Pro-Regenerative Niche after Muscle Injury.

During the process of muscle regeneration post-injury in adults, muscle stem cells (MuSCs) function is facilitated by neighboring cells within the pro-regenerative niche. However, the precise mechanism triggering the initiation of signaling in the pro-regenerative niche remains unknown. Using single-cell RNA sequencing, 14 different muscle cells are comprehensively mapped during the initial stage following injury. Among these, macrophages and fibro-adipogenic progenitor cells (FAPs) exhibit the most pronounced intercellular communication with other cells. In the FAP subclusters, the study identifies an activated FAP phenotype that secretes chemokines, such as CXCL1, CXCL5, CCL2, and CCL7, to recruit macrophages after injury. Il1rl1, encoding the protein of the interleukin-33 (IL-33) receptor, is identified as a highly expressed signature surface marker of the FAP phenotype. Following muscle injury, autocrine IL-33, an alarmin, has been observed to activate quiescent FAPs toward this inflammatory phenotype through the IL1RL1-MAPK/NF-κB signaling pathway. Il1rl1 deficiency results in decreased chemokine expression and recruitment of macrophages, accompanied by impaired muscle regeneration. These findings elucidate a novel mechanism involving the IL-33/IL1RL1 signaling pathway in promoting the activation of FAPs and facilitating muscle regeneration, which can aid the development of therapeutic strategies for muscle-related disorders and injuries.

Transcriptional evidence for transient regulation of muscle regeneration by brown adipose transplant in the rotator cuff.

Chronic rotator cuff (RC) injuries can lead to a degenerative microenvironment that favors chronic inflammation, fibrosis, and fatty infiltration. Recovery of muscle structure and function will ultimately require a complex network of muscle resident cells, including satellite cells, fibro-adipogenic progenitors (FAPs), and immune cells. Recent work suggests that signaling from adipose tissue and progenitors could modulate regeneration and recovery of function, particularly promyogenic signaling from brown or beige adipose (BAT). In this study, we sought to identify cellular targets of BAT signaling during muscle regeneration using a RC BAT transplantation mouse model. Cardiotoxin injured supraspinatus muscle had improved mass at 7 days postsurgery (dps) when transplanted with exogeneous BAT. Transcriptional analysis revealed transplanted BAT modulates FAP signaling early in regeneration likely via crosstalk with immune cells. However, this conferred no long-term benefit as muscle mass and function were not improved at 28 dps. To eliminate the confounding effects of endogenous BAT, we transplanted BAT in the "BAT-free" uncoupling protein-1 diphtheria toxin fragment A (UCP1-DTA) mouse and here found improved muscle contractile function, but not mass at 28 dps. Interestingly, the transplanted BAT increased fatty infiltration in all experimental groups, implying modulation of FAP adipogenesis during regeneration. Thus, we conclude that transplanted BAT modulates FAP signaling early in regeneration, but does not grant long-term benefits.

Curcumin-activated Wnt5a pathway mediates Ca2+ channel opening to affect myoblast differentiation and skeletal muscle regeneration.

BACKGROUND: Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel. METHODS: Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10 weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20 µL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80 mg/L) and fed to the mice (50 mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT-qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining. RESULTS: During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P < 0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.05), and conversely, knockdown of Wnt5a inhibited their expression (P < 0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P < 0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P < 0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated. CONCLUSIONS: Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

Muscle cell-derived Ccl8 is a negative regulator of skeletal muscle regeneration.

Skeletal muscles undergo robust regeneration upon injury, and infiltrating immune cells play a major role in not only clearing damaged tissues but also regulating the myogenic process through secreted cytokines. Chemokine C-C motif ligand 8 (Ccl8), along with Ccl2 and Ccl7, has been reported to mediate inflammatory responses to suppress muscle regeneration. Ccl8 is also expressed by muscle cells, but a role of the muscle cell-derived Ccl8 in myogenesis has not been reported. In this study, we found that knockdown of Ccl8, but not Ccl2 or Ccl7, led to increased differentiation of C2C12 myoblasts. Analysis of existing single-cell transcriptomic datasets revealed that both immune cells and muscle stem cells (MuSCs) in regenerating muscles express Ccl8, with the expression by MuSCs at a much lower level, and that the temporal patterns of Ccl8 expression were different in MuSCs and macrophages. To probe a function of muscle cell-derived Ccl8 in vivo, we utilized a mouse system in which Cas9 was expressed in Pax7+ myogenic progenitor cells (MPCs) and Ccl8 gene editing was induced by AAV9-delivered sgRNA. Depletion of Ccl8 in Pax7+ MPCs resulted in accelerated muscle regeneration after barium chloride-induced injury in both young and middle-aged mice, and intramuscular administration of a recombinant Ccl8 reversed the phenotype. Accelerated regeneration was also observed when Ccl8 was depleted in Myf5+ or MyoD+ MPCs by similar approaches. Our results suggest that muscle cell-derived Ccl8 plays a unique role in regulating the initiation of myogenic differentiation during injury-induced muscle regeneration.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.

Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.

Interleukin 25 promotes muscle regeneration in sarcopenia by regulating macrophage-mediated Sonic Hedgehog signaling.

OBJECTIVE: Sarcopenia manifests as a chronic, low-level inflammation along with multiple inflammatory cells infiltration. Interleukin (IL)-25 can regulate the function of macrophages. However, the specific role and mechanisms through which IL-25 functions in sarcopenia are still not fully understood and require further investigation. METHODS: Aged mice were utilized as sarcopenia models and examined the expression of inflammatory factors. To investigate the effects of IL-25 on sarcopenia, the model mice received IL-25 treatment and underwent in vivo adoptive transfer of IL-25-induced macrophages. Meanwhile, RAW264.7 cells, bone marrow-derived macrophages, satellite cells and C2C12 cells were used in vitro. Shh insufficiency was induced through intramuscular administration of SHH-shRNA adenoviruses. Then, various assays including scratch wound, cell counting kit-8 and Transwell assays, as well as histological staining and molecular biological methods, were conducted. RESULTS: Aged mice exhibited an accelerated decline in muscle strength and mass, along with an increased muscle lipid droplets and macrophage infiltration, and decreased IL-25 levels compared to the young group. IL-25 therapy and the transfer of IL-25-preconditioned macrophages could improve these conditions by promoting M2 macrophage polarization in vivo as well as in vitro. M2 macrophage conditioned medium enhanced satellite cell proliferation and migration, as well as the vitality, migration, and differentiation of C2C12 cells in vitro. Furthermore, IL-25 enhanced Shh expression in macrophages in vitro, and activated the Shh signaling pathway in muscle tissue of aged mice, which could be suppressed by either the inhibitor cyclopamine or Shh knockdown. Mechanistic studies showed that Shh insufficiency suppressed the activation of Akt/mTOR signaling pathway in muscle tissue of aged mice. CONCLUSION: IL-25 promotes the secretion of Shh by M2 macrophages and activates the Shh/Akt/mTOR signaling pathway, which improves symptoms and function in sarcopenia mice. This suggests that IL-25 has potential as a therapeutic agent for treating sarcopenia.